gene identification lab shuba gopal biology department rochester institute of technology...
Post on 20-Dec-2015
215 Views
Preview:
TRANSCRIPT
Gene Identification Lab
Shuba Gopal
Biology Department
Rochester Institute of Technology
sxgsbi@rit.edu
and
Rhys Price Jones
Computer Science Department
Rochester Institute of Technology
rpjavp@rit.edu
Gene Identification involves:
• Locating genes within long segments of genomic sequence.
• Demarcating the initiation and termination sites of genes.
• Extracting the relevant coding region of each gene.
• Identifying a putative function for the coding region.
Outline of Session
• Quick review of genes, transcription and translation
• Gene finding in prokaryotes• Some prokaryotic gene finders• Improving on ORF finding• Gene finding in eukaryotes• Some eukaryotic gene finders
Defining the Gene - 101
• What is the unit we call a gene?- A region of the genome that codes for a functional
component such as an RNA or protein.• We'll focus on protein-coding genes for the remainder of
this session.
• A gene can be further divided into sequence elements with specific functions.
• Genes are regulated and expressed as a result of interactions between sequence elements and the products of other genes.
Schematic of a gene
Control region Coding region 3' UTR
Transcribed region
Promoter,Polymerase binding site,etc.
Poly-adenylationsite5' UTR
Ribosome bindingsite
Finding Genes in Genomes
• Gene = Coding region• What defines a coding region?
- A coding region is the region of the gene that will be translated into protein sequence.
• Is there such a thing as a canonical coding region?
Objective: Identify coding regions computationally from raw genomic sequence data.
Coding Regions as Translation Regions
• Translation utilizes a trinucleotide coding system: codons.
• Translation begins at a start codon.
• Translation ends at a stop codon.
Some Important Codons
• Most organisms use ATG as a start codon.- A few bacteria also GTG and TTG- Regardless of codon used, the first amino acid in
every translated peptide chain is methionine.• However, in most proteins, this methionine is cleaved in
later processing.• So not all proteins have a methionine at the start.
• Almost all organisms use TAG, TGA and TAA as stop codons.- The major exception are the mycoplasmas.
The Degenerate Code
• Of the other 60 triplet combinations, multiple codons may encode the same amino acid.- E.g. TTT and TTC both encode phenylalanine
• Organisms preferentially use some codons over others.
• This is known as codon usage bias.- The age of a gene can be determined in part by the
codons it contains. • Older genes have more consistent codon usage than
genes that have arrived recently in a genome.
Identifying Genes in Genomes
• Organisms utilize a variety of mechanisms to control the transcription and expression of their genes.
• Manipulating gene structure is one such method of control.- Coding regions can be in contiguous segments, or - They may be divided by non-coding regions that can
be selectively processed.
Understanding the Tree of Life
• There are three major branches of the tree:- Bacteria (prokaryote)- Archaea (prokaryote)- Eukaryotes
Coding Regions in Prokaryotes
• In bacteria and archaea, the coding region is in one continuous sequence known as an open reading frame (ORF).
Coding Regions in Prokaryotes
DNA:ATG-GAA-GAG-CAC-CAA-GTC-CGA-TAG
Protein:MET-GLU- GLU -HIS -GLN-VAL-ARG-Stop
Where's Waldo (the Gene)?
• Time for some fun - design your own prokaryote gene finder.
• Follow the lab exercises to identify regions of the E. coli genome that might contain ORFs.
Some Gene Finders in Prokaryotes
• Because the translation region is contiguous in prokaryotes, gene finding focuses primarily on identifying ORFs.- ORF-finder takes a syntactic approach to identifying
putative coding regions. • ORF-finder is available from NCBI.
- GLIMMER 2.0 is a more sophisticated program that attempts to model codon usage, average gene length and other features before identifying putative coding regions.
• GLIMMER 2.0 is available from TIGR.
ORF-Finder
• Approach - Identify every stop codon in the genomic sequence. - Scan upstream to the farthest, in-frame start codon.
• Will locate ORFs that begin with ATG as well as GTG and TTG
- Label this an ORF.
• Output - List all ORFs that exceed a minimum length
constraint.
ORF-Finder
• The black lines represent each of the three reading frames possible on one strand of DNA.
• The gray boxes each represent a putative ORF.
ORF-Finder
• Advantages- Can identify every
possible ORF. - Minimum length
constraint ensures that many false positives are discarded prior to human review.
• Disadvantages- Does not eliminate
overlapping ORFs.- Even with a length
constraint, there are often many false positives.
- Cannot take into account organism-specific idiosyncrasies
ORF-Finder Example
• In this example, there are seven possible ORFs.
• However, only ORF D and G are likely to be coding.
• The others may be eliminated because they are:- Too small
• ORFs A, C and E- Overlap with other ORFs,
• ORFs B, C and F- Have extremely unusual
codon composition.
Glimmer 2.0
• Approach - Build an Interpolated Markov Model (IMM) of the
canonical gene from a set of known genes for the organism of interest.
- The model includes information about: • Average length of coding region • Codon usage bias (which codons are preferentially used) • Evaluates the frequency of occurrence of higher order
combinations of nucleotides from 2 through 8 nucleotide combinations.
Glimmer 2.0
• Output- For each ORF, GLIMMER assigns a likelihood
score or probability that the ORF resembles a known gene.
- High scoring ORFs that overlap significantly with other high scoring ORFs are reported but highlighted.
• GLIMMER 2.0 is reported to be 98% accurate on prokaryotic genomes.
Glimmer 2.0
• Advantages: - Fewer false positives
because ORFs are evaluated for likelihood of coding.
- Organism-specific because model is built on known genes.
- User can modify many parameters during search phase.
• Disadvantages: - Requires
approximately 500+ known genes for proper training.
- Genuine coding regions with unusual codon composition will be eliminated.
- Reported accuracy difficult to reproduce.
Other features of prokaryotic genes
• While the ORF is the defining feature of the coding region, there are other features we can use to identify true coding regions.
• We can improve accuracy by:- Identifying control regions
• Promoters• Ribosome binding sites
- Characterizing composition• CpG islands• Codon usage
Schematic of a gene
Control region Coding region 3' UTR
Transcribed region
Promoter,Polymerase binding site,etc.
Poly-adenylationsite5' UTR
Ribosome bindingsite
Characterizing Promoters
• A promoter is the DNA region upstream of a gene that regulates its expression.- Proteins known as transcription factors bind to
promoter sequences.- Promoter sequences tend to be conserved
sequences (strings) with variable length linker regions.
- Ab initio identification of promoters is difficult computationally.
• A database of known, experimentally characterized promoters is available however.
Ribosome binding sites
• The ribosome binding site (RBS) determines, in part, the efficiency with which a transcript is translated.
• Ribosome binding sites in prokaryotes are relatively short, conserved sequences and have been characterized to some extent.- Eukaryotic ribosome binding sites are more variable
and not as well characterized.- They may also not be conserved from one organism
to another.
Genomic Jeopardy!
• Compare your list of predicted ORFs from the E. coli genome with the verified set from GenBank.- How well did your gene finder perform?- Follow the lab exercises to evaluate your gene
finder.
Characterizing composition
• Codon usage (preferential use of certain codons over others) can be modelled given sufficient data on known genes.- This is part of Glimmer's approach to gene
identification.
• Gene rich regions of the genome tend to be associated with CpG islands.- Regions high in G+C content- Multiple occurrences of CG dinucleotides.- These can be modelled as well.
Summary: Prokaryote Gene Finding
• Prokaryotic coding regions are in one contiguous block known as an open reading frame (ORF).
• Identifying an ORF is just the first step in gene finding.
• The challenge is to discriminate between true coding regions and non-coding ORFs.- Using information from promoter analysis, RBS
identification and codon usage can facilitate this process.
Coding Regions in Eukaryotes
DNA:ATG-GAA-GAG-CAC- *GTTAACACTACGCATACAG* -CAA-GTC-CGA-TAG
Protein:MET-GLU-GLU-HIS-GLN-VAL-ARG-Stop
Gene Finders in Eukaryotes
• Tools for finding genes in eukaryotes - Genie uses information from known genes to guess
what regions of the genome are likely to contain new genes.
- Fgenes is very good at finding exons and reasonably accurate at determining gene structure.
- Genscan is one of the most sophisticated and most accurate.
Genie
• Approach - Apply a pre-built Generalized Hidden Markov Model
(GHMM) of the canonical eukaryotic (mammalian) gene.
- The model includes information about: • Average length of exons and introns. • Compositional information about exons and introns. • A neural-net derived model of splice junctions and
consensus sequences around splice junctions.
- Splice junction information can be further improved by including results of homology searches.
Genie
• Output- Likelihood scores for individual exons - The set of exons predicted to be associated with
any given coding region. - Information regarding alignment of the predicted
coding region to known proteins from homology searching.
• Genie is approximately 60-75% accurate on eukaryotic genomes.
Genie
• Advantages: - Extraneous predicted
exons can be eliminated based on evidence from homology searches.
- Likelihood scores provided for each predicted exon.
• Disadvantages: - No organism-specific
training is possible.- Works best on
mammalian genomes, not other eukaryotes.
- Reliance on homology evidence can result in oversight of novel genes unique to the organism of interest.
Fgenes
• Approach - Identifies putative exons and introns. - Scores each exon and intron based on composition. - Uses dynamic programming to find the highest
scoring path through these exons and introns. • The best-scoring path is constrained by several factors
including that exons must be in frame with each other and ordered sequentially.
Fgenes
• Output- Gene structure derived from best path through
putative exons and introns. - Alternative structures with high scores.
• Fgenes is about 70% accurate in most mammalian genomes.
Fgenes
• Advantages: - Alternative gene
structures are reported.
- Also attempts to identify putative promoter and poly-A sites.
• Disadvantages: - User cannot train
models. - Only human model-
based version is available for unrestricted public use.
Genscan
• Approach- Models for different states (GHMMs)
• State 1 and 2: Exons and Introns - Length - Composition
- State 3: Splice junctions • Weight matrix based array to identify consensus
sequences • Weight matrix to identify promoters, poly-A signals and
other features.
Genscan
• Output- Gene structure - Promoter site - Translation initiation exon - Internal exons - Terminal exon (translation termination) - Poly-adenylation site
• Genscan is 80% accurate on human sequences.
Genscan
• Advantages: - Most accurate of
available tools. - Excellent at identifying
internal and terminal exons
- Provides some assistance in identifying putative promoters
• Disadvantages: - User cannot train
models nor tweak parameters.
- Identification of initial exons is weaker than other kinds of exons.
- Promoter identification can be mis-leading.
Summary for Eukaryote Gene Finding
• Eukaryotic gene structures can be quite complex.
• The best approaches to gene finding in eukaryotes combine probabilistic methods with heuristics to yield reasonable accuracy.- But even in the best case scenario, accuracy is only
about 80%.
top related